Protective effects of low-intensity pulsed ultrasound on mandibular condylar cartilage exposed to mechanical overloading

The aim of this study was to examine the role of low-intensity pulsed ultrasound (LIPUS) exposure in the onset and early progression of temporomandibular joint (TMJ) osteoarthritis (TMJ-OA) induced by mechanical overloading. Fifteen-week-old male Wistar rats were divided into two experimental groups and a control group (n = 5 each). In the experimental groups, both TMJs were subjected to mechanical overloading by forced mouth opening using a jaw-opening device for 3 h/day for 5 continuous days. After mechanical overloading, TMJs in one experimental group were exposed to LIPUS for 20 min/day. After the experiments, mandibles were resected from the rats, and the condyles were processed. The bones were analyzed using high-resolution microcomputed tomography (micro-CT). The resected TMJs were also subjected to histological analysis and immunohistochemical staining. Micro-CT images of the mandibular condyle showed severe subchondral trabecular bone loss in the experimental group with overloading. Treatment with LIPUS after overloading resulted in decreased subchondral trabecular bone resorption. In TMJ sections from the experimental group with overloading, cell-free regions and proteoglycan loss characterized the cartilage degradation; LIPUS exposure restricted these changes in the mandibular condyle. Furthermore, the number of tartrate-resistant acid phosphatase-positive osteoclasts in the mineralized layer of the condylar cartilage increased after mechanical overloading and decreased after LIPUS treatment. Our findings suggest that LIPUS exposure after mechanical TMJ overloading downregulates subchondral trabecular bone resorption and proteoglycan loss in the mandibular condylar cartilage. Thus, it may prove to be protective effects of LIPUS exposure on onset and early progression of TMJ-OA induced by mechanical overloading.


Application of Mechanical Stress
In the experimental groups, both TMJs were subjected to mechanical overloading by forced 116 mouth opening using a jaw-opening device for 3 h/day. The device maintained a mouth opening In the OL+LIPUS group, LIPUS was applied with a modified version of the clinical device,

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Osteotron V (ITO Co., Tokyo, Japan) after mechanical overloading. The ultrasound exposure 124 system was equipped with a circular surface transducer with a cross-sectional area of 5.0 cm 2 .

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The ultrasound head exhibited a mean beam nonuniformity value of 3.6 and an effective radiating 126 area of 4.1 cm 2 . An ultrasound signal was transmitted at a frequency of 1.5 MHz, with a spatial 127 average intensity of 30 mW/cm 2 and a pulse rate of 1:4 (2 ms on and 8 ms off). Both TMJs were 128 exposed to LIPUS for 20 min/day during the 5-day experimental period.

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Microcomputed Tomography (Micro-CT) 131 After the experiments, the rats were sacrificed with an overdose of anesthesia. The animals in 132 the control group were sacrificed after 5 days. The mandibles were resected, and the condyles 133 were carefully separated from the surrounding soft tissues and fixed in 70% ethanol overnight.

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Billeriea, MA) and its analysis software. Briefly, images were acquired at 50 kV and 500 μA.

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During scanning, the samples were tightly covered with plastic wrap to prevent movement and 137 dehydration. Thresholding was applied to the images to segment the bone from the background.

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The resolution of the micro-CT images was 9 μm/pixel.

Statistical Analysis
178 All values are expressed as means and standard deviations. Significant differences in 179 experimental data were analyzed using one-way analysis of variance, followed by the Turkey-

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Kramer test as a post hoc test to examine mean differences at a significance level of 5%.

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The health status of the experimental groups was similar to those in the control group through the 184 experimental period. were higher in the control group than in the OL+LIPUS group, the differences were not significant.  Mankin scores confirmed that overloading caused significant (P < 0.01) changes in structural 224 characteristics that paralleled the progression of OA (Fig 3C). LIPUS exposure after overloading 225 significantly (P < 0.01) lowered the modified Mankin score relative to that for the OL group,

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although the scores were still significantly lower in the control group than in the OL+LIPUS group 227 (P < 0.01). Attenuated toluidine blue staining was observed in the condylar cartilages from the OL 228 group; this reduction in staining was restricted after LIPUS exposure (Fig 3B). The significantly 229 reduced amount of proteoglycans in the condylar cartilage (P < 0.05) after overloading was 230 restored to the level observed for the control group after LIPUS exposure (Fig 3D).

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The number of Col2a1-and aggrecan-positive cells was significantly larger in the control group 263 than in the OL (P < 0.01) and OL+LIPUS (P < 0.05) groups (Fig 5A, B). The OL group showed 264 significantly more MMP9-and MMP13-positive cells than did the control (P < 0.01) and

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OL+LIPUS groups (P < 0.05).    overloading. However, the levels were still significantly lower than those in the control group.

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Micro-CT showed that BV/TV and Tb.Th were significantly greater for mandibular condyles 342 treated with LIPUS after mechanical overloading than for overloaded condyles without LIPUS 343 treatment; moreover, these parameters were almost the same as those for untreated condyles.

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Furthermore, the modified Mankin score for the mandibular condyles from the OL+LIPUS group was significantly lower than that for the condyles from the OL group, although the score for the 346 condyles from the OL+LIPUS group was significantly higher than that for the condyles from the